GnRH
In vertebrates, the hypothalamus and pituitary have well-defined roles in the control of reproduction. GnRH (gonadotropin-releasing hormone) is the central regulatory neurohormone controlling reproduction in all vertebrates. GnRH is a ten amino-acid peptide, synthesized in the hypothalamus and released into the hypophysial portal blood system, directly into the pituitary gland as in the case of teleost fish, or by diffusion as in the case of agnathans. Upon response to external cues (for example as environmental cues such as water temperature) and internal cues GnRH is released and acts at the pituitary gland to stimulate the synthesis and release of the gonadotropins, which in turn travel by systemic circulation to the gonads, thereby regulating steroidogenesis and gametogenesis.
GnRH has been the subject of intense research over many years because of its dual significance for understanding reproductive biology and for developing medical therapies. Aside from its importance in research for understanding reproductive biology, GnRH has many medical and other practical applications including reproductive enhancement and/or contraception in animals and fishes. In fact, GnRH and its analogs are already being used in commercial fish farming to stimulate and regulate sexual maturation and reproduction.
Over the past 15 years or so, a considerable amount of research has been devoted to the effects of GnRH and its analogs on reproduction in fish. Many of the economically important fish do not reproduce spontaneously in captivity. Thus manipulation of their reproductive cycles is crucial to marine aquaculture. Almost all of the research to date has been focused on GnRH-based spawning induction therapy in a number of commercially important species (Zohar et al., 1989). Brood females of salmon and other valuable species will spawn in captivity, but have difficulties in their spawning and the timing of spawning. By implanting a GnRH agonist into a brood female, a fish farmer can ensure that the female will ripen at the proper time, thus preventing potentially costly guesswork. However, while there has been considerable success in achieving high yields in rearing fish, there has been only limited success in the manipulation of the reproductive cycles and spawning of the reared fish. In addition, most of the work to date has focused on or examined the ability of GnRH agonists to induce spawning in females. Few researchers have examined the ability of GnRH antagonists to sterilize male fish, due to its lack of commercial application in aquaculture. However, a new method of sterilization would be very useful in the field of sea lamprey control in the Great Lakes.
During the past few years, the Great Lakes Fisheries Commission (GLFC) has been searching for alternative methods to control sea lamprey populations. In its 1992 Strategic Plan, the GLFC stated that one of its major objectives was to suppress sea lamprey populations to target levels by reduction of the use of lampricides and by development of new control methods by 2010. A compound called Bisazir is currently being used in a sterile-male release program. This compound is extremely hazardous to humans, however, and required a special facility to be constructed at Hammond Bay Biological Station, MI in 1991 for its use. Other chemosterilants that are non-hazardous need to be developed. Although some have suggested inhibiting gonadal development by the negative regulation of GnRH, there have not been any viable methods developed. U.S. Pat. No. 6,210,927 to Zohar, which is incorporated herein in its entirety, for brief mention of inhibition of gonadal development in fish and examples of some uses and applications for seabream GnRH.
Thus, it would be desirable to have a method of sterilizing male sea lampreys, and other fish or animals, using a lamprey or other appropriate species GnRH antagonist.
There are also many potential therapeutic human reproductive applications for GnRH. Since 1971 when the primary structure of mammalian GnRH was determined, over 7,000 analogs to GnRH have been made and tested in hundreds of studies in mammals. So far, the most active synthetic agonists are found to be those with D-amino acid substitution in position 6 of the GnRH decapeptide. The most effective GnRH antagonists to date are those that have substitutions in position 6 as well as substitution of amino acids in positions 1, 2, and 3.
As a result of these studies several mammalian GnRH analogs have been shown to be highly successful and are currently being used for sterilization, conception and other therapeutic and clinical applications. In fact, the clinical application of GnRH analogs as therapeutic drugs generates over 2 billion dollars per year in sales. Hence there is considerable interest in the function of each residue in the GnRH so that analogs can be designed with maximum efficiency as agonists or antagonists to the GnRH receptor, for use as drugs. Furthermore, the responses to GnRH and analogs are different in males compared to females, suggesting that different neuroendocrine mechanisms may be involved.
To date, many analogs have proven useful, but produce undesirable side effects, such as affecting more than just the target. For example, Lupron Depot® which is a GnRH analog and is now one of the leading chemical treatments for advanced prostate cancer and endometriosis in humans has undesirable side effects. For example, continuous treatment of Lupron Depot® results in decreased levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In males, testosterone is reduced to castrate levels. In pre-menopausal females, estrogens are reduced to post-menopausal levels.
Thus, there is still critical information that is needed for understanding the biological activity of these analogs. The potential wider use of GnRH antagonists in humans awaits the availability of potent analogs that do not have the side effects (including high histamine releasing activity) seen with currently-used analogs.
LAMPREY
GnRH has also been studied in several species in the process of researching the evolution of reproductive biology, one of which species is the lamprey. Lampreys and hagfish of the Class Agnatha are of particular importance in understanding endocrinological relationships since they are the modern descendants of the most primitive vertebrates available for study. They represent the oldest lineages of extant vertebrates—which evolved over 550 million years ago. Therefore, the study of lampreys and the characterization of brain and pituitary hormones in lampreys is particularly important for understanding the molecular evolution and functional diversity of reproductive hormones, and can potentially yield valuable insight into human reproductive processes. As noted above, GnRH is the central regulatory neurohormone controlling reproduction in all vertebrates. However, until about 15 years ago, there was little evidence for neuroendocrine control of reproduction in lampreys.
There are approximately 40 species of lampreys that are classified as parasitic or non-parasitic. Lampreys spawn only once in their lifetimes, after which they die. All larval lampreys, called ammocoetes, live in fresh water as borrowing organisms in the bottoms of streams or lakes. In the parasitic sea lamprey, sexual maturation is a seasonal, synchronized process. The sea lampreys begin their lives as fresh water ammocoetes, which are blind, filter feeding larvae. After approximately 5-7 years in freshwater streams, metamorphosis occurs and the ammocoetes become free-swimming, sexually immature lampreys, which migrate to the sea or lakes. During the approximately 15 month-long parasitic sea phase, gametogenesis progresses. After approximately 15 months at sea, lampreys return to freshwater streams and undergo the final maturational processes resulting in mature eggs and sperm, and finally spawning.
As stated above, however, until about 15 years ago, there was a question as to whether there was brain control of reproduction in lampreys. The question of whether there is hypothalamic control over reproduction in lampreys has special significance, because lampreys are modern descendants of the one of the oldest lineages of extant vertebrates and are among the most primitive vertebrates available for study. Thus, the study of lamprey reproduction can shed light on the overall evolution of vertebrate reproduction.
Currently thirteen structures of GnRH have been determined in various vertebrate species and two in invertebrates. They have traditionally been named for the species from which they were first isolated. Table 1 summarizes the various known forms of the GnRH decapeptide. Also, the history of discovery, isolation and characterization of the various known forms of cDNA sequences encoding GnRH precursors is summarized in Table 2 which lists the characterized cDNA's of GnRH precursors.
TABLE 1The 15 known GnRH isoforms, grouped together based on theregions of similarity, with differences from mammalianmGnRH underlined.Table 1-The 15 known GnRH isoforms, grouped together based on the regions of similarity.GnRH12345678910VertebrateMammalpGluHisTrpSerTyrGlyLeuArgProGly-NH2Guinea PigpGluTyrTrpSerTyrGlyValArgProGly-NH2Chicken - IpGluHisTrpSerTyrGlyLeuGlnProGly-NH2RanapGluHisTrpSerTyrGlyLeuTrpProGly-NH2SeabreampGluHisTrpSerTyrGlyLeuSerProGly-NH2SalmonpGluHisTrpSerTyrGlyTrpLeuProGly-NH2MedakapGluHisTrpSerPheGlyLeuSerProGly-NH2CatfishpGluHisTrpSerHisGlyLeuAsnProGly-NH2HerringpGluHisTrpSerHisGlyLeuSerProGly-NH2Chicken - IIpGluHisTrpSerHisGlyTrpTyrProGly-NH2DogfishpGluHisTrpSerHisGlyTrpLeuProGIy-NH2Lamprey - IIIpGluHisTrpSerHisAspTrpLysProGly-NH2Lamprey - IpGluHisTyrSerLeuGluTrpLysProGly-NH2InvertebrateTunicate - IpGluHisTrpSerAspTyrPheLysProGly-NH2Tunicate - IIpGluHisTrpSerLeuCysHisAlaProGly-NH2
The 15 primary structures of GnRH where originally sequenced in pig, mGnRH (Matsuo et al., 1971; Burgus et al., 1972), guinea pig, gpGnRH (Jimenez-Linan et al., 1997), chicken, two forms, chGnRH-I and chGnRH-II (King and Millar, 1982a; King and Millar, 1982b; Miyamoto et al., 1983; Miyamoto et al., 1984), salmon, sGnRH (Sherwood et al. 1983), lamprey, two forms, lGnRH-I and lGnRH-III (Sherwood et al., 1986; Sower et al., 1993), catfish, cfGnRH (Ngamvongchon et al., 1992), dogfish, dGnRH (Lovejoy et al., 1992), herring, hGnRH (Carolsfeld et al., 2000), seabream, sbGnRH (Powell et al., 1994), rana, rGnRH (Yoo et al., 2000), medaka, mdGnRH (Okubo et al., 2000), and tunicate (a protochordate), two forms, tGnRH-I and tGnRH-II (Powell et al., 1996).
TABLE 2The history of discovery, isolation and characterization of the various knownforms of cDNA sequences encoding GnRH precursors.GnRH cDNAs and GenesIsoformOrganismYearReferenceMammalianHuman1984Seeburg et al, NatureNorway Rat1986Adelman et al, PNASMouse1986Mason et al, ScienceNorway Rat1989Bond et al, Mol EndocrinolAfrican Clawed Frog1994Hayes et al, EndoTree Shrew1995White et al Soc NeurosciHaplochromis burtoni1998White et al, Gen Comp EndoJapanese Eel1999Okubo et al, Zool SciBullfrog2001Wang et al, J Exp ZoolSalmonGoldfish1991Bond et al , Mol EndoAtlantic Salmon1992Klungland et al, Mol Cell EndoRainbow Trout1992Alestrom et al, Mol Marine Biol BiotechnolCherry Salmon1992Suzukiet et al, J Mol EndoBrook Trout1992Klungland et al, Mol Cell EndoChinook Salmon1992Klungland et al, Mol Cell EndoRainbow Trout1992Klungland et al, Mol Cell EndoBrown Trout1992Klungland et al, Mol Cell EndoPlainfin Midshipman1995Grober et al, Gen Comp EndoSockeye Salmon1995Coe et al, Mol Cell EndoMedaka2000Okubu et al, Biochem Biophys Res CommunAustralian Bonytongue2001Okubu and Aida, Gen Comp EndoEuropean Sea BassZmora et al (unpublished)ZebrafishTorgersen et al, (unpublished)Verasper moseriAmano (unpublished)Lamprey IIISea Lamprey2002Silver et al, Am ZoolPacific Sea Lamprey2002Silver et al, Am ZoolAustralian Lamprey2002Silver et al, Am ZoolPouched Lamprey2002Silver et al, Am ZoolLamprey ISea Lamprey2001Suzuki et al, J Mol EndoGuinea PigGuinea Pig1997Jimenez-Linan et al,EndoChicken IChicken1993Dunn et al, J Mol EndoRanaFrog2000Yoo et al Mol Cell EndoMedakaMedaka2000Okubu et al, Biochem Brophys Res CommunCatfishAfrican Catfish1994Bogerd et al, Eur J BiochemChicken IIGoldfish1994Bogerd et al, Eur J BiochemHaplochromis burtoni1994White et al, PNASTree Shrew1995White et al, Soc NeurosciRhesus Monkey1996Dong et al, Mol Cell EndoHuman1998White et al, PNASStriped Sea-Bass1998Chow et al, J Mol EndoRhesus Monkey1998White et al, Soc NeurosciHaplochromis burtoni1998White et al, Gen Comp EndoHuman1998White et al, PNASJapanese Eel1999Okubo et al, Zool SciMedaka2000Okubu et al, Biochem Brophys Res CommunAustralian Bonytongue2001Okubo and Aida, Gen Comp EndoBullfrog2001Wang et al, J Exp ZoolVerasper moseriAmano (unpublished)European Sea BassZmora et al (unpublished)Silver-Gray Brushtail PossumLawrence et al (unpublished)Rio Cauca CaecilianEbersole et al, (unpublished)House ShrewWhite et al (unpublished)SeabreamSockeye Salmon1995Ashihara et al, J Mol EndoStriped Sea-Bass1998Chow et al, J Mol EndoHaplochromis burtoni1998White et al, Gen Comp EndoVerasper moseriAmano (unpublished)European Sea BassZmora et al (unpublished)Red Sea BreamOkuzawa (unpublished)
To date, it has been believed that there is only one form of mammalian GnRH that controls the pituitary in mammals. The first GnRH was isolated and characterized from mammals in the early 1970's and is now referred to as mGnRH. However, it is now believed that there are at least two forms of GnRH in all species, which are not just alternative splice variants, but rather are encoded by separate genes (White et al., 1994). The presence of multiple forms of GnRH suggests a functional differentiation, although this has not been characterized.
For example, two main forms of GnRH have been isolated in sea lampreys: lamprey GnRH-I and lamprey GnRH-III. The cDNA (or gene sequence) of lamprey GnRH-I has also been identified, along with cDNA's of eleven of the fifteen known GnRH's in other species. Again, lampreys are studied because they are the most primitive vertebrates for which there are demonstrated functional roles for multiple GnRH neurohormones involved in pituitary-reproductive activity. Thus the study of lamprey can provide insight into higher vertebrate reproduction. Both lamprey GnRH-I and -III have been shown to induce steroidogenesis and spermiation/ovulation in adult sea lampreys (Deragon and Sower, 1994; Gazourian et al., 1997; Sower, 1990; Sower et al., 1993; Sower, 1998).
In studying the various forms of GnRH, which is a ten (10) amino acid protein, the forms most closely related to an ancestral GnRH molecule are most likely the forms present in fishes of ancient origin, for example, lampreys. In all GnRH peptides studied to date, as can be seen from Table 1, certain regions of the molecule have been highly conserved among all species studied, including the NH2-terminal, pGlu1 and Ser4, and the COOH-terminal. The conservation of the NH2- and COOH-termini suggests that these regions are significant for conformation, receptor binding, and resistance to enzymatic degradation, and in receptor-mediated events required for gonadotropin release.
In addition, as can be seen in Table 2, the known cDNA's predict a GnRH consistent with other neuropeptides. The tripartite precursor polypeptide, called prepro-GnRH is synthesized as part of a larger protein which upon post-translational modification yields the mature decapeptide (Klungland et al. 1992). The tripartite prepro-GnRH consists of a leader peptide at the N-terminal hydrophobic signal domain in direct linkage with the GnRH decapeptide; followed by a 3 amino acid dibasic cleavage processing site (GLY-LYS-ARG); and, at the C-terminal end an additional peptide called GnRH associated peptide (GAP). The precursor is processed by cleavage at the dibasic amino acids (LYS-ARG). GnRH and GAP are then stored within the secretory granules until secreted (Wetsel et al., 1991; Endocrinol. 129: 1584-1594).
The mammalian form of GnRH was first isolated form porcine and ovine hypothalamic extracts, giving rise to the popularly held view that only a single form of GnRH is present in all mammals. However, a question that has arisen over the years with respect to mammals is: How does one GnRH differentially regulate the release of two pituitary gonadotropin hormones, LH and FSH? An answer could be found in the fact that, as noted above, in recent years it has been shown that in vertebrates, at least two different forms of GnRH are expressed within the brain, although not necessarily the hypothalamus, of a single species. Generally, where two forms of GnRH have been found, one GnRH is located in the hypothalamus and functions as a neurohormone regulating the pituitary in the control of the gonadotropin release. The second form may have a neurotransmitter or neuromodulatory function and is localized in areas outside the hypothalamus such as in the midbrain regions. In a limited number of mammals a second form of GnRH has been shown to exist, and it is generally extra-hypothalamic. Where two forms of GnRH have been found in a species, it is also believed that separate genes encode for the multiple forms of GnRH (White et al. 1994; Suzuki et al., 2000). In addition, the presence of multiple forms (and locations) of GnRH suggests a functional differentiation (such as differential regulation of FSH and LH), although this has not been characterized.
Based on studies of lampreys and other species in which two forms of GnRH are found, and the high degree of conservation of amino acid sequence between species, study of a known second form of GnRH in one species (for example lampreys) could lead to identification and isolation of a second form of GnRH in other species. If separate genes were found and isolated for multiple forms of GnRH in various species, including primate, and especially human, these findings would have a substantial impact on our understanding of the release of gonadotropins and would be of great value in clinical studies and practical applications.
In addition, the elucidation of the nucleotide sequence of the cDNA's and/or genes of GnRH and other brain hormones in the lamprey is necessary in order to answer questions concerning both comparative analysis of species and the molecular evolution of neuroendocrine hormones in vertebrates. Using such knowledge, in both humans and other species, new GnRH analogs could be developed that may not have the side effects produced with the GnRH analogs currently used in various applications. Study of a novel GnRH in lampreys, and the evolutionary insights yielded therefrom, could lead to discovery of a novel hypothalamic GnRH in mammals which could lead to the formation of new, useful GnRH analogs.
Thus, despite the knowledge of GnRH to date, there remains a need in both marine aquaculture and human medicine for greater knowledge of GnRH and the evolution of neuroendocrine hormones. This knowledge could be used to produce more effective analogs which could better manipulate reproduction in fish, and which could more effectively be used in human therapy, including reproductive and cancer therapy among others.